Benzodiazepine (BZD) receptors [1]
- Benzodiazepine (BZD) receptors (partial agonist-like binding in BALB/c mice, no affinity data specified) [2]
- Benzodiazepine (BZD) receptors (mediating allopregnanolone's anxiolytic action blockade) [3]
- Benzodiazepine (BZD) receptors and non-NMDA receptors (interactions with non-NMDA antagonists in seizure models) [4]
- Benzodiazepine (BZD) receptors (blocking anxiety in ethanol-withdrawn rats) [5]

The behavioral, neurological, and electrophysiological effects of benzodiazepine agonists and inverse agonists are neutralized or reversed by flumazenil through its interaction with central benzodiazepine receptors. Hepatic encephalopathy can benefit from flumazenil in certain cases, however until well planned clinical trials can be carried out, hepatic encephalopathy must be treated as an exploratory indication. Although flumazenil can restore the drowsiness brought on by benzodiazepines either by alone or in conjunction with other medications, it shouldn't be taken if cyclic antidepressant toxicity is suspected [1]. In the raised plus maze and light/dark test, flumazenil (1 mg/kg) significantly reduces anxiety in BALB/c mice [2]. In rats, the decrease in allopregnanolone can be successfully stopped by flumazenil (10 mg/kg) [3]. In mice, the anticonvulsant and side effects of diazepam are countered by flumazenil (5–20 mg/kg), but not by GYKI 52466. The anticonvulsant activity of NBQX was somewhat diminished by flumazenil in the MES model, but not in the PTZ trial [4]. Reduced open-arm time and open-arm entry percentage are the outcomes of long-term ethanol treatment because flumazenil (3.0 mg/kg) inhibits withdrawal alterations [5].

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Flumazenil reversed the sedative, hypnotic, anxiolytic, anticonvulsant, and muscle relaxant effects of benzodiazepines in various animal models and human studies [1]
- Flumazenil induced partial agonist-like effects (reduced locomotor activity, increased punished responding in the Vogel conflict test) in BALB/c mice but not in C57BL/6 mice [2]
- Flumazenil (10 mg/kg, i.p.) completely blocked the anxiolytic action of allopregnanolone (5 mg/kg, i.p.) in the elevated plus-maze test in mice [3]
- Flumazenil (10 mg/kg, i.p.) antagonized the anticonvulsant effects of diazepam against pentylenetetrazol-induced seizures in mice, but did not affect the anticonvulsant actions of NBQX or GYKI 52466 [4]
- Flumazenil (1-10 mg/kg, i.p.) dose-dependently blocked the anxiety-like behavior (increased entries into open arms of elevated plus-maze) in rats undergoing ethanol withdrawal [5]

Radioligand binding assays were performed to evaluate the affinity of Flumazenil for benzodiazepine receptors in brain membrane preparations. Membranes were incubated with tritiated benzodiazepine ligands in the presence or absence of Flumazenil, followed by filtration and scintillation counting to measure specific binding inhibition [1]
- Receptor binding studies in mouse brain homogenates showed that Flumazenil displaced radiolabeled benzodiazepine ligands, with differential binding profiles between BALB/c and C57BL/6 mouse strains [2]

Mice (BALB/c and C57BL/6 strains) were used to assess behavioral effects of Flumazenil. The drug was dissolved in a suitable vehicle and administered intraperitoneally at doses ranging from 0.1 to 10 mg/kg. Behavioral tests included locomotor activity monitoring and the Vogel conflict test, performed 30 minutes after drug administration [2]
- Mice were treated with Flumazenil via intraperitoneal injection at a dose of 10 mg/kg, 15 minutes prior to allopregnanolone administration (5 mg/kg, i.p.). The elevated plus-maze test was conducted 30 minutes after allopregnanolone administration to evaluate anxiolytic activity [3]
- Mice were pretreated with Flumazenil (10 mg/kg, i.p.) 30 minutes before administration of diazepam, NBQX, or GYKI 52466. Seizures were induced by pentylenetetrazol, and seizure severity and latency were recorded for 30 minutes post-induction [4]
- Rats were rendered ethanol-dependent by chronic ethanol vapor exposure. After ethanol withdrawal, Flumazenil was administered intraperitoneally at doses of 1, 3, and 10 mg/kg. Anxiety-like behavior was evaluated using the elevated plus-maze test 30 minutes after drug administration [5]
- Human subjects with benzodiazepine overdose or therapeutic benzodiazepine effects were administered Flumazenil via intravenous infusion at a rate of 0.2 mg/min (total dose up to 1 mg) to reverse benzodiazepine-induced sedation and respiratory depression [1]

Absorption, Distribution and Excretion
Flumazenil is completely metabolized (99%). The elimination of the radiolabeled drug is essentially completed within 72 hours, with 90% to 95% of the radioactive material appearing in urine and 5% to 10% in feces. Dosage: 0.9 to 1.1 L/kg 1 L/hr/kg [Healthy volunteers receive 1 mg infusion over 5 minutes] Metabolism/Metabolites Hepatic metabolism. Flumazenil is completely metabolized (99%). The major metabolites of flumazenil detected in urine are deethylated free acid and its glucuronide conjugates. Biological half-life Initial distribution half-life is 4 to 11 minutes, and terminal half-life is 40 to 80 minutes. The half-life is prolonged to 1.3 hours in patients with moderate hepatic impairment and to 2.4 hours in patients with severe hepatic impairment. Compared with adults, the elimination half-life of pediatric patients was more variable, averaging 40 minutes (range: 20 to 75 minutes).

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Flumazenil is rapidly absorbed after intravenous administration, with a volume of distribution in the human body of approximately 0.6–1.0 L/kg. The elimination half-life is 0.7–1.3 hours [1]
- Flumazenil is extensively metabolized in the liver through oxidation and conjugation reactions, with less than 1% of the original drug excreted unchanged in the urine [1]
- Due to significant first-pass metabolism, the oral bioavailability of flumazenil is low (approximately 16%) [1]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation There is currently no information regarding the clinical use of flumazenil during lactation. Since flumazenil is not absorbed orally, it is unlikely to have adverse effects on breastfed infants. If the mother needs to take flumazenil, there is no need to stop breastfeeding. The drug has a half-life of 54 minutes, therefore stopping breastfeeding 4 to 5 hours after administration minimizes drug transfer to the infant. ◉ Effects on Breastfed Infants As of the revision date, no relevant published information was found. ◉ Effects on Lactation and Breast Milk As of the revision date, no relevant published information was found. Protein Binding The protein binding rate is approximately 50%, primarily (66%) bound to albumin. Protein binding is reduced in patients with cirrhosis. Flumazenil has low toxicity in humans. Adverse reactions are mild and transient, including nausea, vomiting, anxiety, and seizures (rare, mainly seen in patients with epilepsy or benzodiazepine dependence) [1]
- Flumazenil has a plasma protein binding rate of approximately 40-50% [1]

[1]. Flumazenil: a benzodiazepine antagonist. Clin Pharm. 1993 Sep;12(9):641-56; quiz 699-701.

[2]. Flumazenil induces benzodiazepine partial agonist-like effects in BALB/c but not C57BL/6 mice. Psychopharmacology (Berl). 2000 Jan;148(1):24-32.

[3]. Flumazenil blocks the anxiolytic action of allopregnanolone. Eur J Pharmacol. 1995 Jul 25;281(1):113-5.

[4]. Effects of the non-NMDA antagonists NBQX and the 2,3-benzodiazepine GYKI 52466 on different seizure types in mice: comparison with diazepam and interactions with flumazenil. Br J Pharmacol. 1994 Dec;113(4):1349-57.

[5]. Flumazenil blockade of anxiety following ethanol withdrawal in rats. Psychopharmacology (Berl). 1997 Jun;131(4):354-60.

Flumazenil is an organic heterocyclic compound with the chemical name 5,6-dihydro-4H-imidazo[1,5-a][1,4]benzodiazepine, in which the 3, 5, 6, and 8 positions are substituted with ethoxycarbonyl, methyl, oxo, and fluorine atoms, respectively. It can be used as an antidote for benzodiazepine overdose. Flumazenil acts as a γ-aminobutyric acid (GABA) antagonist and is also an antidote for benzodiazepine poisoning. It is an ethyl ester, an organofluorine compound, and an imidazobenzodiazepine. Flumazenil is an imidazobenzodiazepine derivative and a potent benzodiazepine receptor antagonist. It competitively inhibits the activity of the benzodiazepine recognition site on the GABA/benzodiazepine receptor complex, thereby reversing the effects of benzodiazepines on the central nervous system. Flumazenil is a benzodiazepine antagonist. Flumazenil is an imidazobenzodiazepine derivative that effectively reverses benzodiazepine-induced activity. Flumazenil antagonizes the benzodiazepine binding site of the GABA/benzodiazepine receptor complex in the central nervous system (CNS), thereby preventing chloride channel opening and inhibiting neuronal hyperpolarization. Therefore, flumazenil can reverse the sedation, psychomotor disturbances, amnesia, and hypoventilation caused by benzodiazepines in a dose-dependent manner. It is a potent benzodiazepine receptor antagonist. Because it can reverse the sedative and other effects of benzodiazepines, it is considered an antidote for benzodiazepine overdose. Indications: For complete or partial reversal of the sedative effects of benzodiazepines, suitable for induction and/or maintenance of general anesthesia with benzodiazepines, and for sedation with benzodiazepines during diagnostic and therapeutic procedures. In addition, it can be used as an adjunct therapy to treat benzodiazepine overdose, in conjunction with appropriate supportive and symptomatic treatment.
FDA Label

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Mechanism of Action
Flumazenil is an imidazobenzodiazepine derivative and belongs to the benzodiazepine antagonist class. It exerts its effect by competitively inhibiting the benzodiazepine binding site on the GABA/benzodiazepine receptor complex. In some animal models, flumazenil is a weak partial agonist, but in humans it has little or no agonist activity.

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Pharmacodynamics
Flumazenil antagonizes the central nervous system effects of benzodiazepines, but it does not antagonize the central nervous system effects of drugs that affect GABAergic neurons through pathways other than benzodiazepine receptors (including ethanol, barbiturates, or general anesthetics), nor does it reverse the effects of opioids.

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Flumazenil (Ro 15-1788) is a selective competitive benzodiazepine receptor antagonist used clinically to reverse the effects of benzodiazepine overdose and terminate conscious sedation [1]
-The partial agonist-like effect of flumazenil in BALB/c mice suggests strain-dependent differences in benzodiazepine receptor signaling or regulation [2]
-Flumazenil blocks the anxiolytic effect of allogeneinolone, indicating that allogeneinolone exerts its anxiolytic effect through benzodiazepine receptor regulation [3]
-Flumazenil does not interact with non-NMDA glutamate receptors because it failed to affect the anticonvulsant activity of NBQX or GYKI 52466 [4]
-The anxiolytic blocking effect of flumazenil in ethanol withdrawal rats suggests that benzodiazepine receptor activation is involved in ethanol withdrawal-induced anxiety [5]

C15H14FN3O3

303.29

303.101

78755-81-4

3373

White to off-white solid powder

1.4±0.1 g/cm3

528.0±50.0 °C at 760 mmHg

201-203°C

273.1±30.1 °C

0.0±1.4 mmHg at 25°C

1.634

0.67

0

5

3

22

461

0

OFBIFZUFASYYRE-UHFFFAOYSA-N

InChI=1S/C15H14FN3O3/c1-3-22-15(21)13-12-7-18(2)14(20)10-6-9(16)4-5-11(10)19(12)8-17-13/h4-6,8H,3,7H2,1-2H3

ethyl 8-fluoro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2 mg/mL (6.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2 mg/mL (6.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2 mg/mL (6.59 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)